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Content archived on 2024-05-27

Mechanistic insights into protein degradation by Lon, a AAA+ protease

Objective

Background: AAA+ proteases are ubiquitous molecular machines that execute regulated protein degradation in cells. Their activity is necessary for regulation of many cellular processes, as well as to clear the cell of misfolded, potentially harmful proteins. Failure to combat protein unfolding in unicellular organisms often leads to reduced viability, and in humans, accumulation of misfolded proteins may result in prion diseases, cancer and neurodegenerative disorders. Conserved in bacteria and eukaryotic organelles, the major enzyme responsible for degrading damaged proteins is a hexameric AAA+ protease known as Lon. Despite its central role in most living organisms, the mechanism of substrate degradation by this protease is poorly understood. Objectives & Work Plan: To reveal the detailed molecular mechanism of protein degradation by Lon and related AAA+ proteases by studying the Lon protease of Escherichia coli as a model system. The research will exploit the fact that Lon is the only AAA+ protease for which a natural protein inhibitor – PinA, a T4-bacteriophage protein – is known. Genetic screens will be conducted for the isolation of Lon mutants with improved mechanistic properties that result in faster substrate processing, and for active mutants that are resistant to PinA inhibition (aim 1). To obtain insight into the mechanistic properties of the mutants, biochemical characterization will be carried out using native and unfolded protein substrates, a wide variety of peptide substrates, and sensitive assays for the detection of binding and degradation (aim 2). To solve the 3D structure of Lon, primary crystallization screens will be set-up to optimize the crystallization conditions so as to obtain crystals that diffract at high resolution (aim 3). Relevance to Work Programme: The reintegration grant will aid me to establish an independent laboratory conducting top-level protein research and thereby to obtain a permanent position in university in the ERA.

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Topic(s)

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Call for proposal

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FP7-PEOPLE-2009-RG
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Funding Scheme

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MC-IRG - International Re-integration Grants (IRG)

Coordinator

BEN-GURION UNIVERSITY OF THE NEGEV
EU contribution
€ 100 000,00
Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

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